SUMMARY The development of inhibitory antibodies (inhibitors) against FVIII is not only a severe and important complication of protein replacement therapy, but also a major concern in gene therapy of hemophilia A. Generation of such inhibitors might potentially preclude gene therapy for hemophilia A. In this project, we propose to investigate a novel gene therapy approach that will provide therapeutic FVIII protein and induce immune tolerance for hemophilia A and hemophilia A with inhibitors based on the hypothesis that targeting the production of FVIII to platelets that activate at the site where FVIII is needed could overcome the presence of inhibitory antibodies. In addition, targeting neo-protein FVIII expression and storage in the ?-granules of platelets that contain immunomosuppresive molecule, transforming growth factor beta-1, and that aged platelets undergo apoptosis and phagocytosis would generate immunoregulatory milieu, promoting antigen- specific immunologic tolerance. We have developed a clinically translatable gene therapy protocol for hemophilia A using lentiviral gene delivery of the FVIII expression cassette under control of the platelet-specific ?IIb promoter (2bF8) to hematopoietic stem cells resulting in FVIII expression in platelets. Our previous studies using animal models have demonstrated that platelet targeted FVIII expression results in FVIII storage together with its carrier protein VWF in platelet ?-granules and that platelets-FVIII retains efficacy even in the presence of inhibitors with no overt thrombotic risk. Our further studies show that 2bF8 lentiviral gene delivery to hematopoietic stem cells can not only restore hemostasis in hemophilia A mice but also induce immune tolerance through peripheral clonal deletion of CD4 T cells and induction of antigen-specific regulatory T cells. We found that VWF is essential for platelet gene therapy of HA with inhibitors. In the current application, we propose to develop optimal preconditioning regimen for platelet gene therapy for hemophilia A and hemophilia A with inhibitors. We will elucidate how VWF/FVIII interaction impacts platelet-FVIII delivery and FVIII immune responses in platelet-specific FVIII gene therapy. We will explore the potential underlying mechanisms by which the unique peripheral tolerance is processed after 2bF8 gene therapy. These studies should help us to further understand the biological characteristics of 2bF8 gene therapy, with the potential to develop a safe curative gene therapy approach that can not only provide therapeutic protein, but also induce the antigen-specific immune tolerance for the clinical treatment of HA patients and patients with inhibitors, as well as non-hereditary hemophilic patients with acquired inhibitory antibodies that can also have life-threatening clinical bleeding.